Abstract

The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.

Overview of the experiment. (A) Schematic showing the rodent CST originating from the pyramidal CSNs in layer V of the sensorimotor cortex, decussating at the spinomedullary junction, forming the main dCST and the dlCST and vCST minor components. Rats received six unilateral viral vector injections into the sensorimotor cortex to transduce the CSNs and bilateral C1/C2 intraspinal injections of the retrograde tracer Fast Blue to label the CSNs. (B) Image showing transduced, retrogradely labelled CSNs. (C) Image showing GFP-positive CST fibres in the contralateral dCST of the cervical spinal cord.

The mean area of transduction per section and the mean GFP intensity per neuron for each viral vector. (A) Quantification of the mean area of transduction per section was determined for each viral vector. AAV1 transduced a significantly larger area of cortex per section than the other viral vectors. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests * P < 0.05, n = 3/group. Asterisk indicates a key significant comparison; the complete set of significant comparisons is described in Results. (B) The mean GFP intensity per neuron was measured by outlining the soma of 20 randomly selected neurons per section for each rat. The mean GFP intensity per neuron was not significantly different between the viral vectors. Values represent mean and SEM, analysis was performed using one way ANOVA, P > 0.05, n = 3/group.

Transduction of CSNs by seven AAV serotypes and an integration-deficient lentiviral vector. (A) Quantification of transduced CSNs. The CSNs were labelled using the retrograde tracer Fast Blue. The number of GFP and Fast Blue positive neurons was counted and the mean number of transduced CSNs plotted for each viral vector. AAV1 transduced a significantly higher number CSNs than the other viral vectors. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests *** P < 0.001, * P < 0.05, n = 3/group. (B) Quantification of the percentage of CSNs transduced relative to both the total number of retrogradely traced CSNs and the number of retrogradely traced CSNs in the area of transduction. Compared to the other viral vectors AAV1 transduced a significantly higher percentage of CSNs relative to the total number of CSNs or the number of CSNs within the area of transduction. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests *** P < 0.001, * P < 0.05, n = 3/group. Asterisks show a subset of key significant comparisons; the complete set of significant comparisons is described in Results. (C) High magnification images of AAV1 transduced CST projecting pyramidal CSNs from different animals. Scale bars: 50 μm.

GFP-positive fibres in the dCST of the cervical spinal cord. (A) Quantification of transduced fibres in the dCST at spinal level C1. GFP-positive fibres were counted and the mean number plotted for each viral vector. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests * P < 0.05, n = 3/group. (B) When the viral vectors were compared using the Pearson correlation coefficient a significant, positive correlation (R2 = 0.88) was observed between the number of transduced CSNs and the number of GFP-positive CST fibres. (C) Images of GFP-positive fibres in the contralateral dCST at C1 from rats transduced with the AAV serotypes or lentiviral vector. GFP-positive axon collaterals can be seen entering the grey matter in the images of AAV5 and 6. Scale bar: 150 μm.

The viral vectors did not generate a microglial response. (A) An Iba1 stained cortex from an AAV1 transduced rat. No observable difference in Iba1 immunoreactivity was seen between the two hemispheres. The 800 μm × 900 μm boxes used to quantification the number of microglia are shown. Scale bar: 500 μm. (B) Higher-magnification image of the 800 μm × 900 μm box from the uninjected hemisphere in (A). Scale bar: 200 μm. (C) Higher-magnification image of the 800 μm × 900 μm box from the injected hemisphere in (A). (D) Higher-magnification image of the needle track in (A, arrow) showing enhanced Iba1 staining along the needle track as indicated by the arrow. (E) Quantification of the relative number of microglia within the area of transduction. The number of Iba1 positive cells in an 800 μm × 900 μm box placed over each hemisphere was counted, the relative number of microglia, expressed as a percentage of the contralateral hemisphere was then calculated and plotted for each viral vector. Values represent mean and SEM, analysis was performed using one way ANOVA P > 0.05, n = 3/group.